Amylin is a newly discovered 37-amino acid peptide which was first isolated from islet amyloid found in the pancreases of human patients with non-insulin dependent diabetes mellitus (NIDDM), also known as Type II diabetes. (Cooper et al., Biochem. Biophys. Acta. 1014:247-258 (1989); Cooper et al., Proc. Nat'l. Acad. Sci. USA 84:8628-8632 (1987)). Islet .beta.-cells cosecrete amylin and insulin in response to glucose and amino acids. (Ogawa et al., J. Clin. Invest. 85:973-976 (1990)). We have discovered that amylin and insulin together regulate carbohydrate metabolism in vitro and in vivo. Administration of amylin can prevent the primary occurrence of insulin-induced hypoglycemia in normal people. Inappropriate production of amylin can lead to diabetes. For instance, it has been reported that animals which model the NIDDM disease state demonstrate over-production of amylin at the transcriptional level. (Jamal et al., J. Endocrinol. 126:425-429 (1990).
Insulin-dependent diabetes mellitus (IDDM) patients suffer from autoimmune destruction of pancreatic .beta.-cells resulting in the reduction of insulin production which, in turn, causes hyperglycemia. A serious side-effect of insulin therapy for IDDM patients is hypoglycemia which is made more severe by the absence of amylin due to autoimmune destruction of the islet .beta.-cells. Evidence has been collected which confirms differences in the circulating blood levels of amylin in Type I diabetics, persons with obesity and impaired glucose tolerance, and normal people. (Ludvik, et al., Diabetes 39 Supp. 1, 116A (1990); Harttner, E., et al. Lancet, 1990, 854; Harttner, E., et al., Lab. Med. 14:229 (1990)). Because amylin has been reported to play a pivotal role in diabetes and other insulin resistant states, there has been a need to generate synthetic amylin in order to be able to further investigate its physiological properties.
Fully active human amylin contains an internal disulfide bond and a C-terminal amide group. Although the amino acid sequence is reported to be well conserved in a number of species, the physical properties of individual amylins have been found to vary depending on the source. (Cooper, G. J. S., et al., Proc. Nat'l. Acad. Sci. USA 84:8626-8632 (1987); Leffert, J. D. et al. Proc. Nat'l. Acad. Sci. USA 86:5738-5742 (1989); Nishi, M. et al. Proc. Nat'l. Acad. Sci. USA 86:3127-3130 (1989)). For instance, amyloid formation, which is characterized by an abnormal aggregation of peptide, is observed with human and feline amylin but not with the rodent analogs. (Nishi, M., et al., Proc. Nat'l. Acad. Sci. USA 86:3127-3130 (1989)).
Although the biological activity of amylin has been examined with natural material isolated from human pancreatic islet amyloid (Leighton, B. and Cooper, G. S. S., Nature, 335:632-635 (1988)), most studies have used amylin produced by solid phase peptide synthesis. There exist several publications which report a failure to demonstrate certain previously observed or predicted biological activities of amylin (Bretherton-Watt, D., et al., Diabetologia 33:115-117 (1990); Ghatei, M. A., et al. J. Endocrinol. 124:R9-R11(1990)). Attempts at synthesis of human amylin by synthetic methods have also resulted in preparations which are widely variable with respect to chemical purity as well. Such preparations have been contaminated by deletion peptides formed during the process of chemical synthesis. In order to be able to perform further studies examining the biological activities of amylin and reliable quantitative studies with synthetic human amylin, sufficient amounts of peptide of adequate purity and biological activity and which may be standardized (with respect to both purity and activity) are needed.
Experiments designed to examine the biological activities of human amylin in which previously available preparations of peptide were employed must include an evaluation of the chemical and biological variability of those preparations. The contamination of these amylin preparations with peptides of unknown, and possibility deleterious, activities will impede in vivo studies with these peptides, especially in humans.